Osteocytes (OCY) are intrinsically three-dimensional (3D), mature bone cells encased in 3D mineralized extracellular bone matrix. Recent studies indicate the critical roles of osteocytes in detecting mechanical signals and maintaining skeleton integrity. These roles have significant clinical implications, such as in the etiology of osteoporosis or new pharmaceutical targets for osteoporosis treatment. A novel 3D trabecular bone explant co-culture model for osteocyte-osteoblast mechanobiology in this proposal allows for live osteocytes to be surrounded by their native extracellular matrix environment and to interconnect with osteoblasts (OB) through intercellular processes in the canaliculi channels. We propose to use this novel 3D trabecular bone co-culture model to test a central scientific hypothesis that that dynamic deformational loading induces OCYs to send anabolic signals to OBs to promote bone formation through intraceluar calcium [Ca2+]i oscillations in osteocytic network, followed by prostaglandin E2 (PGE2) production/secretion via gap junctions/hemi-channels to OBs. We will test the following working hypotheses: Hypothesis H1: PGE2 production, changes in bone formation, and elastic modulus of 3D bovine trabecular bone explants with seeded primary bovine OBs depend on calmodulin kinase (CaMK) dependent Ca2+ oscillations in OCYs. Hypothesis H2: PGE2 production, changes in bone formation, and elastic modulus of 3D bovine trabecular bone explants with seeded primary bovine OBs depend on the gap junctions/hemi-channels connexin 43 (Cx43) in OCYs. Hypothesis H3: Bone formation response of OBs seeded in 3D trabecular bone explants under dynamic deformational loading and changes in elastic modulus of trabecular bone depend on PGE2 pathway. With this new co-culture system of trabecular bone explants, the interaction between osteocytes and osteoblasts under mechanical loading can be investigated in vitro under conditions that are more physiologically relevant than previously possible: (1) both osteocytes and osteoblasts are included and positioned in their native 3D trabecular bone environment when subjected to dynamic deformational loading; (2) functional bone formation and elastic modulus of trabecular bone will be assessed in vitro, linking important factors in osteocyte-osteoblast mechanotransduction to bone functions; (3) selectively manipulating biochemical pathways in OCYs and OBs independently with sophisticated molecular biology technique, which cannot be achieved in vivo, and (4) micromechanical environments surrounding osteocytes and/or osteoblasts will be quantified, respectively, using specimen specific finite element models. New insights will be gained regarding cellular and molecular mechanisms of bone cell mechanotransduction and will contribute to our general understanding of the etiology of osteoporosis, and may lead to therapeutic interventions aimed at the mitigation or treatment of osteoporosis.